It is estimated that over 50% of all economic losses in weaned pigs are due to Escherichia coli infections, causing either diarrhea or edema disease. In addition to the E. coli strains responsible for disease in pigs, other E. coli, as well as Salmonella, strains also colonize the intestinal tract of pigs. Many of these strains are of major concern for human food safety. The U.S. Centers for Disease Control and Prevention (CDC) estimates that in the year 2000 over 1.4 million people suffered and more than 600 died, in this country, from foodborne disease caused by Salmonella and E. coli 0157:H7. The costs attributed to these diseases were approximately $3.1 billion.
The bacterial strains considered primarily responsible for E. coli infections in pigs, F4 (K88) and F18, are not well controlled by traditional prophylactic antibiotic treatments. With worldwide concerns over the use of prophylactic antibiotics in animal agriculture, the development of alternatives to conventional antibiotics is urgently needed to protect swine from these E. coli infections.
Probiotics have been explored as one alternative to the use of conventional antibiotics. A “probiotic” strategy is one that employs the use of microflora to reduce pathogenic bacteria (including food-borne pathogens) in the gut. Probiotic techniques involve the introduction of a healthy microbial population to the gastrointestinal (GI) tract, or providing a limiting nutrient, sometimes termed a “prebiotic”, that allows an existing commensal microbial population to expand its role in the gastrointestinal tract. The addition of a non-pathogenic microbial culture to the intestinal tract of food animals in order to reduce colonization or decrease populations of pathogenic bacteria in the gastrointestinal tract is referred to as “competitive exclusion”.
Competitive exclusion cultures may be composed of a single strain, several strains, or even several species of microorganisms. Depending on the stage of production, or more specifically, the maturity of the gut, the goal of this culture can be the exclusion of pathogens from the naïve gut of a neonatal animal, or the displacement of an already established pathogenic bacterial population.
Some bacteria produce antimicrobial protein compounds (traditional antibiotics, as well as bacteriocins, or colicins) in order to eliminate competitive bacteria, and have therefore shown additional promise for their use in competitive exclusion products. Protein antibiotics are attractive alternatives to conventional antibiotics used in animal feed, since they are not absorbed intact by the animal and, therefore, leave no antibiotic residues in the meat. Additionally, bacteriocins have the potential for very favorable regulatory status by the U.S. Food and Drug Administration. Nisin, a bacteriocin, is generally regarded as safe for use as a food additive for its antimicrobial properties. The possibility of an effective antibiotic alternative being regulated as a food additive, rather than as a new animal drug, is further incentive for bacteriocin research among the animal health/feed industries.
Colicins are classified as either pore-forming or nuclease colicins based on their mode of bacteriocidal activity, and are further categorized based on their mode of membrane integration in sensitive bacteria. Members of both classes have been shown effective against gram-negative bacteria of concern for animal health and human food safety, such as E. coli and Salmonella strains, and therefore hold promise for use as alternatives to conventional antibiotics in animal diets.
Colicins are a class of bacteriocins produced by, and effective against E. coli and closely related members of the family Enterobacteriaceae. Pore-forming colicins are between 387 and 626 amino acids in length, and provide their antibacterial effect by crossing the outer membrane, spanning the periplasm, and inserting into the bacterial inner cell membrane to form voltage-dependent ion channels. The ion leakage caused by these channels uncouples energy expenditures from growth, causing death in cellular targeted bacteria. Nuclease colicins kill sensitive cells by non-specific degradation of DNA or specific cleavage of rRNA.
Shiga toxin producing E. coli strains, such as 0157:H7, which present serious human food safety concerns, have also been shown to be sensitive to colicins. Doyle et al., U.S. Pat. No. 5,965,128, discloses the use of colicin producing E. coli as probiotics in cattle to reduce E. coli 0157:H7 shedding. Further, Lyon et al., U.S. Pat. No. 5,549,895, discloses the use of naturally produced colicins for inhibiting E. coli 0157:H7 and other Escherichia species, as well as Shigella species in food products, on carcasses, and on hard surfaces as a sanitizer.
Although colicins have shown potential as alternatives to conventional antibiotics in animal feed, it would not be cost effective to purify this protein from naturally occurring colicin producing E. coli strains, nor to include the levels of these bacteria necessary to obtain an antimicrobial effect in the feed.
It is therefore a primary objective of the present invention to produce alternatives to conventional antibiotics for use in the animal feed industry.
It is a further objective of the present invention to produce recombinant colicins in a yeast expression system.
It is a further objective of the present invention to produce recombinant colicins in a plasmid expression system.
It is still a further objective of the present invention to produce recombinant colicins that are effective against pathogenic bacteria.
It is yet a further objective of the present invention to produce recombinant colicins that may be used as a probiotic culture.
It is yet a further objective of the present invention to produce recombinant colicins that are effective against E. coli and Salmonella strains of importance to human food safety.
It is a further objective of the present invention to produce recombinant colicins that are effective against pathogenic bacteria that is cost effective.
The method and means of accomplishing each of the above objectives as well as others will become apparent from the detailed description of the invention which follows hereafter.